Complex evaluation of human monocyte‐derived dendritic cells for cancer immunotherapy

Dendritic cell (DC) immunotherapy is capable of generating tumour‐specific immune responses. Different maturation strategies were previously tested to obtain DC capable of anti‐cancer responses in vitro, usually with limited clinical benefit. Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application. DC were generated from monocytes using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4, pulsed with whole tumour cell lysate and then matured with one of five selected maturation strategies or cultured without additional maturation stimulus. DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens. We were able to demonstrate extensive variability among different maturation strategies currently used in DC immunotherapeutic protocols that may at least partially explain limited clinical benefit of some clinical trials with such DC. We identified DC matured with interferon‐γ and lipopolysaccharide as the most attractive candidate for future clinical trials in cancer immunotherapy.     

Natural antiviral compound silvestrol modulates human monocyte‐derived macrophages and dendritic cells

Outbreaks of infections with viruses like Sars‐CoV‐2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona‐, Ebola‐, Zika‐, Picorna‐, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down‐regulated several pro‐ and anti‐inflammatory cytokines (IL‐6, IL‐8, IL‐10, CCL2, CCL18) and increased TNF‐α during differentiation and activation of M1‐macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti‐inflammatory potential of M2‐macrophages by increasing expression of anti‐inflammatory surface markers CD206, TREM2 and reducing release of pro‐inflammatory IL‐8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down‐regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.     

Differential phagocytosis of Leishmania mexicana promastigotes and amastigotes by monocyte‐derived dendritic cells

Leishmania species are dimorphic protozoan parasites that live and replicate in the gut of sand flies as promastigotes or in mammalian hosts as amastigotes. Different immune cells, including DCs, and receptors differ in their involvement in phagocytosis of promastigotes and amastigotes and in recognition of different Leishmania species. In the case of L. mexicana, differences in phagocytosis of promastigotes and amastigotes by DCs and participation of C‐type lectin receptors (CLRs) have not been established. In the present study, flow cytometry and confocal microscopy were used to investigate the phagocytosis by monocyte‐derived dendritic cells (moDCs) of L. mexicana promastigotes and amastigotes in the presence or absence of immune serum during various periods of time. Blocking antibodies against mannose receptors and DC‐SIGN were used to explore the participation of these receptors in the phagocytosis of L. mexicana by moDC. The major differences in interactions of L. mexicana promastigotes and amastigotes with moDC were found to occur within the first 3 hr, during which phagocytosis of promastigotes predominated as compared with opsonization of promastigotes and amastigotes. However, after 6 hr of incubation, opsonized promastigotes were preferentially phagocytosed as compared with unopsonized promastigotes and amastigotes and after 24 hr of incubation there were no differences in the phagocytosis of promastigotes and amastigotes. Finally, after 3 hr incubation, DC‐SIGN was involved in the phagocytosis of promastigotes, but not of amastigotes.     

Malaria hemozoin modulates susceptibility of immature monocyte‐derived dendritic cells to HIV‐1 infection by inducing a mature‐like phenotype

Together, Plasmodium falciparum (P. falciparum) and HIV‐1 infections cause more than four million deaths a year. There is still limited information about the putative impact of the malaria pigment hemozoin (HZ) on the dissemination of HIV‐1. As so, we propose a premise where HZ present in human dendritic cells (DCs) could modulate HIV‐1 transfer to CD4⁺ T cells. We report here that HZ promotes transmission of HIV‐1 by immature monocyte‐derived DCs (iMDDCs). Moreover, we noted that in the presence of HZ, iMDDCs were less permissive to productive HIV‐1 infection. The HZ‐dependent modulation of the interaction between iMDDCs and HIV‐1 seems to be partly due to a decreased expression of CCR5 and also to the induction of a more mature phenotype as proven by microscopy and flow cytometry analyses. Therefore, exposure of iMDDCs to malaria pigments provokes their maturation rendering them more potent to trans‐infect CD4⁺ T cells with HIV‐1.     

Role of Kir2.1 in human monocyte‐derived foam cell maturation

The role of K⁺ channels in macrophage immunomodulation has been well‐established. However, it remains unclear whether K⁺ channels are involved in the lipid uptake of macrophages. The expression and function of the inward rectifier potassium channel (Kir2.1, KCNJ2) in Human acute monocytic leukemia cell line (THP‐1) cells and human monocytes derived macrophages (HMDMs) were investigated using RT‐PCR and western blotting, and patch clamp technique. The expression of scavenger receptors in THP‐1–derived macrophages was detected using western blotting. Expressions of Kir2.1 mRNA and protein in HMDMs were significantly decreased by 60% (P < 0.05) and 90% (P < 0.001) on macrophage maturation, but overexpressed by approximately 1.3 (P > 0.05) and 3.8 times (P = 0.001) after foam cell formation respectively. Concurrently, the Kir2.1 peak current density in HMDMs, mature macrophages and foam cells, measured at −150 mV, were −22.61 ± 2.1 pA/pF, −7.88 ± 0.60 pA/pF and −13.39 ± 0.80 pA/pF respectively (P < 0.05). In association with an up‐regulation of Kir2.1 in foam cells, the SR‐A protein level was significantly increased by over 1.5 times compared with macrophages (P < 0.05). THP‐1 cells contained much less lipids upon Kir2.1 knockdown and cholesterol ester/total cholesterol ratio was 29.46 ± 2.01% (P < 0.05), and the SR‐BI protein level was increased by over 6.2 times, compared to that of macrophages (P < 0.001). Kir2.1 may participate in macrophage maturation and differentiation, and play a key role in lipid uptake and foam cell formation through modulating the expression of scavenger receptors.     

Highly pathogenic avian influenza H5N1 virus induces cytokine dysregulation with suppressed maturation of chicken monocyte‐derived dendritic cells

One of the major causes of death in highly pathogenic avian influenza virus (HPAIV) infection in chickens is acute induction of pro‐inflammatory cytokines (cytokine storm), which leads to severe pathology and acute mortality. DCs and respiratory tract macrophages are the major antigen presenting cells that are exposed to mucosal pathogens. We hypothesized that chicken DCs are a major target for induction of cytokine dysregulation by H5N1 HPAIV. It was found that infection of chicken peripheral blood monocyte‐derived dendritic cells (chMoDCs) with H5N1 HPAIV produces high titers of progeny virus with more rounding and cytotoxicity than with H9N2 LPAIV. Expression of maturation markers (CD40, CD80 and CD83) was weaker in both H5N1 and H9N2 groups than in a LPS control group. INF‐α, ‐β and ‐γ were significantly upregulated in the H5N1 group. Pro‐inflammatory cytokines (IL‐1β, TNF‐α and IL‐18) were highly upregulated in early mid (IL‐1), and late (IL‐6) phases of H5N1 virus infection. IL‐8 (CXCLi2) mRNA expression was significantly stronger in the H5N1 group from 6 hr of infection. TLR3, 7, 15 and 21 were upregulated 24 hr after infection by H5N1 virus compared with H9N2 virus, with maximum expression of TLR 3 mRNA. Similarly, greater H5N1 virus‐induced apoptotic cell death and cytotoxicity, as measured by terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling and lactate dehydrogenase assays, respectively, were found. Thus, both H5N1 and H9N2 viruses evade the host immune system by inducing impairment of chMoDCs maturation and enhancing cytokine dysregulation in H5N1 HPAIV‐infected cells.     

Modulation of concanavalin A stimulation of hamster lymphoid cells by lithium chloride.

Addition of LiCl (1–25 mM) to serum-free cultures of MHA hamster thymocytes, lymph node cells, or splenocytes stimulated with concanavalin A had a biphasic effect on [³H]thymidine incorporation. These concentrations of LiCl enhanced stimulation of [³H]thymidine incorporation by suboptimal levels of concanavalin A but inhibited stimulation of optimal and supraoptimal concentrations of concanavalin A. This effect was specific for Li⁺ since it was not observed when similar concentrations of Na⁺, K⁺, or Mg²⁺ were added to cultures stimulated by concanavalin A. The inhibitory effect of LiCl on concanavalin A stimulation was not reversed by addition of Na⁺, Ca²⁺, Mg²⁺, or Ca²⁺ + Mg²⁺ to the cultures. Significant reversal of LiCl inhibition of stimulation was observed when KCl was added to the cultures. However none of the ions tested blocked the Li-induced enhancement of [³H]thymidine incorporation in the presence of suboptimal concentrations of concanavalin A.     

Functionality of insect‐cell‐derived colorectal cancer vaccine candidate protein EpCAM‐Fc in human dendritic cells

The baculovirus–insect cell expression system is widely used to produce recombinant proteins for various biomedical applications. Our previous study demonstrated that EpCAM, a colorectal cancer vaccine candidate protein, can be expressed in the baculovirus–insect cell expression system. However, its functionality (the ability to elicit an immune response), which is important for its possible use as a colorectal cancer vaccine for immunotherapy, still needed to be confirmed. In this study, we examined the ability of recombinant EpCAM to induce maturation of immature dendritic cells (DCs) derived from CD34⁺ cells isolated from human umbilical cord blood. We demonstrated that EpCAM induces the expression of four DC maturation markers: CD80, CD83, CD86 and MHC II. These results suggest that EpCAM produced in the baculovirus–insect cell expression system is functional in terms of its ability to trigger maturation of human DCs.     

Malaria‐derived hemozoin exerts early modulatory effects on the phenotype and maturation of human dendritic cells

Plasmodium falciparum (P. falciparum)‐induced effects on the phenotype of human dendritic cells (DC) could contribute to poor induction of long‐lasting protective immunity against malaria. DC ability to present antigens to naïve T cells, thus initiating adaptive immune responses depends on complex switches in chemokine receptors, production of soluble mediators and expression of molecules enabling antigen‐presentation and maturation. To examine the cellular basis of these processes in the context of malaria, we performed detailed analysis of early events following exposure of human monocyte‐derived DC to natural hemozoin (nHZ) and the synthetic analog of its heme core, β‐hematin. DC exposed to either molecule produced high levels of the inflammatory chemokine MCP‐1, showed continuous high expression of the inflammatory chemokine receptor CCR5, no upregulation of the lymphoid homing receptor CCR7 and no cytoskeletal actin redistribution with loss of podosomes. DC partially matured as indicated by increased expression of major histocompatibility complex (MHC) class II and CD86 following nHZ and β‐hematin exposure, however there was a lack in expression of the maturation marker CD83 following nHZ but not β‐hematin exposure. Overall our data demonstrate that exposure to nHZ partially impairs the capacity of DC to mature, an effect in part differential to β‐hematin.     

Human monocyte‐derived macrophages spontaneously differentiated in vitro show distinct phenotypes

Tissue macrophages are resident phagocytes that acquire specific phenotypes according to the microenvironment. Morphological and functional heterogeneity has been evidenced in different homeostatic and pathological conditions. Indeed, the nature of macrophage subsets may have either harmful or beneficial functions in disease progression/resolution. Therefore the possibility to pharmacologically manipulate heterogeneity represents a relevant challenge. Since human tissue macrophages are not easily obtained, various in vitro models are currently used that do not adequately reflect the heterogeneity and plasticity of tissue macrophages. We had previously reported that two dominant and distinct macrophage morphotypes co‐exist in the same culture of human monocytes spontaneously differentiated for 7 days in autologous serum. The present study was aimed to the phenotypic characterization of these morphotypes, that is, round‐ and spindle‐shaped. We observed that, besides substantial differences in cytoskeleton architecture, round monocyte‐derived macrophages (MDMs) showed higher lipid content, increased macropinocytosis/efferocytosis capacity, and overexpression of CD163, interleukin (IL)‐10, and transforming growth factor (TGF) β2. Conversely, spindle MDMs exhibited enhanced respiratory burst and higher expression of the chemokine (C‐C motif) ligands 18 and 24 (CCL18 and CCL24). Overall, round MDMs show functional traits reminiscent of the non‐inflammatory and reparative M2 phenotype, whereas spindle MDMs exhibit a pro‐inflammatory profile and express genes driving lymphocyte activation and eosinophil recruitment. MDMs obtained in the culture condition herein described represent a valuable model to disentangle and manipulate the functional heterogeneity of tissue macrophages that has been disclosed in scenarios spanning from inflammatory and wounding responses to atherosclerotic lesions. J. Cell. Physiol. 228: 1464–1472, 2013. © 2012 Wiley Periodicals, Inc.     

Extremely low‐frequency electromagnetic fields affect the immune response of monocyte‐derived macrophages to pathogens

This study aimed to determine the effect of extremely low‐frequency electromagnetic fields (ELF‐EMF) on the physiological response of phagocytes to an infectious agent. THP‐1 cells (human monocytic leukemia cell line) were cultured and 50 Hz, 1 mT EMF was applied for 4–6 h to cells induced with Staphylococcus aureus or interferon gamma/lipopolysaccharide (IFγ/LPS). Alterations in nitric oxide (NO), inducible nitric oxide synthase (iNOS) levels, heat shock protein 70 levels (hsp70), cGMP levels, caspase‐9 activation, and the growth rate of S. aureus were determined. The growth curve of exposed bacteria was lower than the control. Field application increased NO levels. The increase was more prominent for S. aureus‐induced cells and appeared earlier than the increase in cells without field application. However, a slight decrease was observed in iNOS levels. Increased cGMP levels in response to field application were closely correlated with increased NO levels. ELF‐EMF alone caused increased hsp70 levels in a time‐dependent manner. When cells were induced with S. aureus or IFγ/LPS, field application produced higher levels of hsp70. ELF‐EMF suppressed caspase‐9 activation by a small extent. These data confirm that ELF‐EMF affects bacterial growth and the response of the immune system to bacterial challenges, suggesting that ELF‐EMF could be exploited for beneficial uses. Bioelectromagnetics 31:603–612, 2010. © 2010 Wiley‐Liss, Inc.     

Thymus‐derived regulatory T cells restrain pro‐inflammatory Th1 responses by downregulating CD70 on dendritic cells

The severity and intensity of autoimmune disease in immune dysregulation, polyendocrinopathy, enteropathy, X‐linked (IPEX) patients and in scurfy mice emphasize the critical role played by thymus‐derived regulatory T cells (tTregs) in maintaining peripheral immune tolerance. However, although tTregs are critical to prevent lethal autoimmunity and excessive inflammatory responses, their suppressive mechanism remains elusive. Here, we demonstrate that tTregs selectively inhibit CD27/CD70‐dependent Th1 priming, while leaving the IL‐12‐dependent pathway unaffected. Immunized mice depleted of tTregs showed an increased response of IFN‐γ‐secreting CD4⁺ T cells that was strictly reliant on a functional CD27/CD70 pathway. In vitro studies revealed that tTregs downregulate CD70 from the plasma membrane of dendritic cells (DCs) in a CD27‐dependent manner. CD70 downregulation required contact between Tregs and DCs and resulted in endocytosis of CD27 and CD70 into the DC. These findings reveal a novel mechanism by which tTregs can maintain tolerance or prevent excessive, proinflammatory Th1 responses.     

Platelet‐derived growth factor promotes the proliferation of human umbilical cord‐derived mesenchymal stem cells

This study was designed to investigate the effect of platelet‐derived growth factor (PDGF) on the proliferation of human umbilical cord mesenchymal stem cells (UC‐MSCs) and further explore the mechanism of PDGF in promoting the proliferation of UC‐MSCs. The human UC‐MSCs were treated with different concentrations of PDGF, and the effects were evaluated by counting the cell number, the cell viability, the expression of PDGF receptors analyzed by RT‐PCR, and the detection of the gene expression of cell proliferation, cell cycle and pluripotency, and Brdu assay by immunofluorescent staining and Quantitative real‐time (QRT‐PCR). The results showed that PDGF could promote the proliferation of UC‐MSCs in vitro in a dose‐dependent way, and 10 to 50 ng/ml PDGF had a significant proliferation effect on UC‐MSCs; the most obvious concentration was 50 ng/ml. Significant inhibition on the proliferation of UC‐MSCs was observed when the concentration of PDGF was higher than 100 ng/ml, and all cells died when the concentration reached 200 ng/ml PDGF. The PDGF‐treated cells had stronger proliferation and antiapoptotic capacity than the control group by Brdu staining. The expression of the proliferation‐related genes C‐MYC, PCNA and TERT and cell cycle–related genes cyclin A, cyclin 1 and CDK2 were up‐regulated in PDGF medium compared with control. However, pluripotent gene OCT4 was not significantly different between cells cultured in PDGF and cells analyzed by immunofluorescence and QRT‐PCR. The PDGF could promote the proliferation of human UC‐MSCs in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Induction of hepatocyte‐like cells from human umbilical cord‐derived mesenchymal stem cells by defined microRNAs

Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl₄‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

Qualitative differences in T‐cell activation by dendritic cell‐derived extracellular vesicle subtypes

Exosomes, nano‐sized secreted extracellular vesicles (EVs), are actively studied for their diagnostic and therapeutic potential. In particular, exosomes secreted by dendritic cells (DCs) have been shown to carry MHC‐peptide complexes allowing efficient activation of T lymphocytes, thus displaying potential as promoters of adaptive immune responses. DCs also secrete other types of EVs of different size, subcellular origin and protein composition, whose immune capacities have not been yet compared to those of exosomes. Here, we show that large EVs (lEVs) released by human DCs are as efficient as small EVs (sEVs), including exosomes, to induce CD4⁺ T‐cell activation in vitro. When released by immature DCs, however, lEVs and sEVs differ in their capacity to orient T helper (Th) cell responses, the former favouring secretion of Th2 cytokines, whereas the latter promote Th1 cytokine secretion (IFN‐γ). Upon DC maturation, however, these functional differences are abolished, and all EVs become able to induce IFN‐γ. Our results highlight the need to comprehensively compare the functionalities of EV subtypes in all patho/physiological systems where exosomes are claimed to perform critical roles.